Improving diagnostic and prognostic accuracy of myeloid neoplasms (MNs) by next generation sequencing.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e18567-e18567
Author(s):  
Candice Schwartz ◽  
Jason Nathaniel Macklis ◽  
Lela Buckingham ◽  
Jamile M. Shammo
Keyword(s):  
Rna Splicing ◽  
Somatic Mutations ◽  
Prognostic Significance ◽  
Primary Myelofibrosis ◽  
Definitive Diagnosis ◽  
Driver Mutations ◽  
Or Management ◽  
Panel Testing ◽  
Gene Panel Testing ◽  
The Impact

e18567 Background: Molecular aberrations in MNs have been well described. Driver mutations such as JAK2, MPL, and CALR, are pathognomonic for MNs, whereas other somatic mutations are less specific but have prognostic significance. Multi-gene panel testing for known somatic mutations has been utilized for diagnostic and prognostic purposes, and testing for somatic mutations to identify clonal hematopoiesis has been adopted by the WHO 2016 classification of myeloid malignancies. We sought to assess the impact of testing for somatic mutations by NGS on diagnosis and management of pts with MNs. Methods: We employed a myeloid panel (MP) of 40 commonly mutated genes involving RNA splicing, chromatin remodeling, and signaling pathways. Testing was performed on ptswho presented to the clinic between 2/2015 and 12/2016. Initial diagnosis and rationale for testing (diagnostic or prognostic) were recorded. We then determined whether the MP resulted in a change in diagnosis, prognosis, or management. Results: 55 pts with a known or suspected MN had a MP performed.Diagnoses at presentation were: MDS (27), MF (8), MPN-U (8), MDS/MPN (4), multiple diagnoses (2), and no definitive diagnosis of MN (6). 87% (48/55) of pts had at least one somatic mutation.In 13 pts (23.6%) the MP led to a definitive diagnosis or a change in diagnosis. For example, 2 pts initially diagnosed with MPN-U were diagnosed with CNL after detection of CSF3R mutation. All 4 pts initially diagnosed with MDS with fibrosis were subsequently diagnosed with primary myelofibrosis; 3 had a MPL mutation and 1 had a CALR mutation. Management was altered in 12 pts (21.8%) and prognosis was changed in 11 pts (20.0%). For example, 2 pts were treated with a JAK-2 inhibitor and 2 pts with low risk MDS were referred for transplant evaluation due to the presence of a TP53mutation. Conclusions: Our study confirms that panel testing meaningfully improves diagnostic accuracy and provides prognostic value. In total, the MP resulted in a change in diagnosis, prognosis, or management in 43.6% (24/55) of cases. Confirmation of these observations merits prospective evaluation for a larger number of pts.

scholarly journals Clonal approaches to understanding the impact of mutations on hematologic disease development

Blood ◽  
2019 ◽  
Vol 133 (13) ◽  
pp. 1436-1445 ◽  
Author(s):  
Jyoti Nangalia ◽  
Emily Mitchell ◽  
Anthony R. Green
Keyword(s):  
Somatic Mutations ◽  
Clonal Selection ◽  
Single Cells ◽  
Driver Mutations ◽  
Great Promise ◽  
Tumor Evolution ◽  
Disease Development ◽  
Hematopoietic Tissue ◽  
The Impact

Abstract Interrogation of hematopoietic tissue at the clonal level has a rich history spanning over 50 years, and has provided critical insights into both normal and malignant hematopoiesis. Characterization of chromosomes identified some of the first genetic links to cancer with the discovery of chromosomal translocations in association with many hematological neoplasms. The unique accessibility of hematopoietic tissue and the ability to clonally expand hematopoietic progenitors in vitro has provided fundamental insights into the cellular hierarchy of normal hematopoiesis, as well as the functional impact of driver mutations in disease. Transplantation assays in murine models have enabled cellular assessment of the functional consequences of somatic mutations in vivo. Most recently, next-generation sequencing–based assays have shown great promise in allowing multi-“omic” characterization of single cells. Here, we review how clonal approaches have advanced our understanding of disease development, focusing on the acquisition of somatic mutations, clonal selection, driver mutation cooperation, and tumor evolution.

scholarly journals Clonal haematopoiesis in chronic ischaemic heart failure: prognostic role of clone size for DNMT3A- and TET2-driver gene mutations

2020 ◽  
Author(s):  
Birgit Assmus ◽  
Sebastian Cremer ◽  
Klara Kirschbaum ◽  
David Culmann ◽  
Katharina Kiefer ◽  
...  
Keyword(s):  
Heart Failure ◽  
Somatic Mutations ◽  
Prognostic Significance ◽  
Gene Mutations ◽  
Amplicon Sequencing ◽  
Driver Mutations ◽  
Driver Gene ◽  
Roc Curve Analysis ◽  
Driver Genes ◽  
Clone Size

Abstract Aims Somatic mutations of the epigenetic regulators DNMT3A and TET2 causing clonal expansion of haematopoietic cells (clonal haematopoiesis; CH) were shown to be associated with poor prognosis in chronic ischaemic heart failure (CHF). The aim of our analysis was to define a threshold of variant allele frequency (VAF) for the prognostic significance of CH in CHF. Methods and results We analysed bone marrow and peripheral blood-derived cells from 419 patients with CHF by error-corrected amplicon sequencing. Cut-off VAFs were optimized by maximizing sensitivity plus specificity from a time-dependent receiver operating characteristic (ROC) curve analysis from censored data. 56.2% of patients were carriers of a DNMT3A- (N = 173) or a TET2- (N = 113) mutation with a VAF >0.5%, with 59 patients harbouring mutations in both genes. Survival ROC analyses revealed an optimized cut-off value of 0.73% for TET2- and 1.15% for DNMT3A-CH-driver mutations. Five-year-mortality was 18% in patients without any detected DNMT3A- or TET2 mutation (VAF < 0.5%), 29% with only one DNMT3A- or TET2-CH-driver mutations above the respective cut-off level and 42% in patients harbouring both DNMT3A- and TET2-CH-driver mutations above the respective cut-off levels. In carriers of a DNMT3A mutation with VAF ≥ 1.15%, 5-year mortality was 31%, compared with 18% mortality in those with VAF < 1.15% (P = 0.048). Likewise, in patients with TET2 mutations, 5-year mortality was 32% with VAF ≥ 0.73%, compared with 19% mortality with VAF < 0.73% (P = 0.029). Conclusion The present study defines novel threshold levels for clone size caused by acquired somatic mutations in the CH-driver genes DNMT3A and TET2 that are associated with worse outcome in patients with CHF.

New Insight Into the Biology, Risk Stratification, and Targeted Treatment of Myelodysplastic Syndromes

2017 ◽  
pp. 480-494
Author(s):  
Mintallah Haider ◽  
Eric J. Duncavage ◽  
Khalid F. Afaneh ◽  
Rafael Bejar ◽  
Alan F. List
Keyword(s):  
Risk Stratification ◽  
Myelodysplastic Syndromes ◽  
Rna Splicing ◽  
Somatic Mutations ◽  
Prognostic Significance ◽  
Gene Mutations ◽  
International Prognostic Scoring System ◽  
Prognostic Assessment ◽  
Sequencing Part ◽  
Gene Panels

In myelodysplastic syndromes (MDS), somatic mutations occur in five major categories: RNA splicing, DNA methylation, activated cell signaling, myeloid transcription factors, and chromatin modifiers. Although many MDS cases harbor more than one somatic mutation, in general, there is mutual exclusivity of mutated genes within a class. In addition to the prognostic significance of individual somatic mutations, more somatic mutations in MDS have been associated with poor prognosis. Prognostic assessment remains a critical component of the personalization of care for patient with MDS because treatment is highly risk adapted. Multiple methods for risk stratification are available with the revised International Prognostic Scoring System (IPSS-R), currently considered the gold standard. Increasing access to myeloid gene panels and greater evidence for the diagnostic and predictive value of somatic mutations will soon make sequencing part of the standard evaluation of patients with MDS. In the absence of formal guidelines for their prognostic use, well-validated mutations can still refine estimates of risk made with the IPSS-R. Not only are somatic gene mutations advantageous in understanding the biology of MDS and prognosis, they also offer potential as biomarkers and targets for the treatment of patients with MDS. Examples include deletion 5q, spliceosome complex gene mutations, and TP53 mutations.

Prognostic Impact of Bone Marrow Fibrosis in Primary Myelofibrosis: A Study of Agimm Group on 540 Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 351-351 ◽  
Author(s):  
Paola Guglielmelli ◽  
Giada Rotunno ◽  
Annalisa Pacilli ◽  
Elisa Rumi ◽  
Vittorio Rosti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Multivariate Analysis ◽  
High Risk ◽  
Board Of Directors ◽  
Research Funding ◽  
Primary Myelofibrosis ◽  
Driver Mutations ◽  
Prognostic Impact ◽  
Advisory Committees ◽  
The Impact

Abstract Background. The prognostic significance of bone marrow (BM) fibrosis grade in pts with primary myelofibrosis (PMF) is debated. A fibrosis grade greater than 1 was associated with a 2-fold higher risk of death compared with pts with early/prefibrotic MF (grade 0) [Thiele J, Ann Hematol 2006]. Recent data suggest that more accurate prediction of survival is achieved when fibrosis grade is added to IPSS [Verner C, Blood 2008; Giannelli U, Mod Pathol 2012]. Aim. To analyze the prognostic impact of fibrosis in diagnostic BM samples of 540 WHO-2008 diagnosed PMF pts with extensive clinical and molecular information collected in 6 Italian centers belonging to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative). Methods. The clinical variables assessed were those previously identified as prognostically relevant in the IPSS score. Published methods were used to screen mutations of JAK2, MPL, CALR, EZH2, ASXL1, IDH1/2 and SRSF2. European consensus scoring system was used to grade fibrosis (on a scale of MF-0 to MF-3). The prognostic value of fibrosis with regard to overall survival (OS) was estimated by Kaplan-Meier method and Cox regression. Results. Pts' median age was 61y; median follow-up 3.7y; median OS 10.5y; 184 pts (34.1%) died. IPSS risk category: low 33.7%, Int-1 27.7%, Int-2 19.1%, High-risk 19.5%. Mutational rate: JAK2 V617F 62.6%, CALR 20.7% (type-1/1-like 77.7%, type2/2-like-2 21.4%), MPL W515 5.9%; 62 (11.5%) were triple negative (TN). 171 pts (31.7%) were High-Molecular Risk (HMR) category (Vannucchi AM, Leukemia 2013); mutation rate: EZH2 7.2%, ASXL1 22.2%, IDH1-2 2.4%, SRSF2 8.3%. According to fibrosis grading, 50 pts were MF-0 (9.3%), 180 MF-1 (33.3%), 196 MF-2 (36.3%), 114 MF-3 (21.1%). Compared with both MF-0 and MF-1, MF-2 and MF-3 pts presented more frequently constitutional symptoms (P<.0001), larger splenomegaly (P<.0001), greater risk of developing anemia (P<.0001) or thrombocytopenia (P=.003). We found a significant association (P<.0001) between IPSS higher/Int-2 risk categories and MF-2 and -3 (20.5% and 37.8%, respectively, vs 14.8% and 6.0% for MF-0 and -1). There was no correlation between fibrosis grade and phenotypic driver mutations; in particular, TN pts were equally distributed among MF fibrosis grades (10%, 10.6%, 14.3% and 8.8% from MF-0 to -3, respectively). Conversely, the frequency of HMR pts increased progressively according to fibrosis grade: 8 pts MF-0 (16%), 46 MF-1 (25.6%), 66 MF-2 (33.7%) and 51 MF-3 (44.7%) (P<.0001). In particular, we found a significant association between fibrosis grade and ASXL1 (12%, 15%, 23.5% and 36% from MF-0 to -3; P<.0001) and EZH2 (2%, 3.9%, 8.2%, 13.2%; P=.01) mutations. Also, pts with 2 or more HMR mutated genes were preferentially MF-2 or -3 ( 0%, 4.4% 10.2% and 10.5% from MF-0 to -3; P=.001). Median OS was significantly shorter in pts with MF-2 (OS 6.7y, HR 7.3, IC95% 2.7-20.0; P<.0001) and MF-3 (OS 7.2y, HR 8.7, IC95% 3.1-24.2; P<.0001) compared with MF-1 (14.7y; HR 3.9, IC95% 1.4-10.9, P=.008) and MF-0 (P<.0001) used as reference group (OS not reached) (Figure). Excluding MF-0, MF-2 and -3 maintained negative prognostic impact with HR 1.9 (1.3-2.6; P=.001) and 2.2 (1.5-3.3; P<.0001) respectively vs MF-1. The impact of fibrosis on OS was maintained when analysis was restricted to younger (≤65y) pts. In multivariate analysis using the individual IPSS variables, grade MF-2 and -3 were independently predictive of survival (HR 3.9 (1.4-10.8), and HR 4.2 (1.5-12.0), respectively, P=.008 for both). The negative impact on survival of MF-2/-3 was maintained regardless of IPSS category, HMR status, number of HMR mutated genes and driver mutations, included as covariates (Table). In low, Int-1 and Int-2, but not high-risk IPSS categories, MF-2/-3 associated with reduced survival (P<.03). Conclusions. Overall, these results indicate that higher grades (MF-2 and MF-3) of fibrosis correlate with defined clinical and molecular variables and independently negatively impact on OS in PMF, suggesting the opportunity to explore its value in the setting of clinical and molecular prognostic scores for PMF. Table. Multivariate Analysis Variables HR 95% CI P value HMR status 2.4 1.5-3.7 <.0001 HMR≥2mutations 4.3 2.8-6.4 .009 IPSS scoring Int1 2.9 1.6-5.1 <.0001 Int2 10.0 5.6-17.7 <.0001 High 9.7 5.5-17.2 <.0001 Driver mutations CALR type2 3.4 1.3-8.6 .010 JAK2/MPL 2.4 1.4-4.3 .003 TN 4.5 2.3-8.8 <.0001 Fibrosis MF-2/MF-3 3.8 1.4-10.6 .010 Figure 1. Figure 1. Disclosures Passamonti: Novartis: Consultancy, Honoraria, Speakers Bureau. Barbui:Novartis: Speakers Bureau. Vannucchi:Shire: Speakers Bureau; Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals The Impact of Somatic Mutations upon the Response to Combination Therapy with Ruxolitinib and Interferon in MPN Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3589-3589
Author(s):  
Vibe Skov ◽  
Anders Lindholm Sørensen ◽  
Trine Alma Knudsen ◽  
Mads Emil Bjørn ◽  
Christina Ellervik ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Treatment Response ◽  
Allele Frequency ◽  
Somatic Mutations ◽  
Triple Negative ◽  
Median Number ◽  
Post Treatment ◽  
Driver Mutations ◽  
Molecular Response ◽  
The Impact

Abstract Introduction: The Philadelphia-negative myeloproliferative neoplasms (MPN) are associated with driver mutations in JAK2, CALR, and MPL genes. Non-driver mutations affect disease progression and treatment response. Combination therapy with pegylated interferon-alpha2 (IFN) and ruxolitinib has recently been shown to induce hematologic and molecular responses in patients (pts) with MPN. We studied 25 pts by targeted next generation sequencing (NGS) of 42 genes and investigated the impact of somatic mutations (mut) on treatment response. Methods: Twenty-five MPN pts with polycythemia vera (PV) (n=16, all JAK2V617F) and myelofibrosis (MF) (n=9, JAK2V617F (6), CALR (1), MPL (1), triple-negative (1)) participated in the study. NGS was performed on the Illumina platform on DNA from peripheral blood at baseline and after 24 months of combination therapy. Data were analyzed using Biomedical Genomics workbench and VarSeq. Variants with coverage &lt;100x, variant allele frequency (VAF) &lt;1%, introns and germline variants, and SNPs with minor allele frequency &gt;1% were excluded. A mut with VAF &lt;1% in either a pre- or post-treatment sample was retained if VAF was &gt;1% in the paired sample. However, a mut with a number of mutated reads below 7 was considered absent. Only pathogenic mut were reported. Statistical analysis was done using either chi-square test or Wilcoxon signed rank/rank sum test in R 4.0.2. A p-value &lt;0.05 was considered significant. The ELN and IWG-MRT response criteria were used. Results: At baseline and 24 months, PV-pts had a mean number of mut of 2.3 and 2.4, and MF-pts 2.2 and 3.0, respectively. In all pts, 0 and 1 (0/4%) patient had no mut, 10 and 7 (40/28%) had 1 mut, 10 and 9 (40/36%) had 2 mut, and 5 and 8 (20/32%) had ≥ 3 mut at baseline and 24 months, respectively. In PV, 1 (6%) achieved CR and 3 (19%) achieved PR and 12 (75%) achieved NR. In MF, 4 (44%) achieved CR and 1 (11%) achieved PR and 4 either NR, PD, SD or CI. There was no association between pts achieving CR/PR or NR and median number of mut at baseline (CR/PR: 1, range 1-3) or (NR: 2, range 1-8), (p=0.28). Excluding driver mut, there was still no significant association. In all JAK2V617F positive pts, the median JAK2V617F allele burden (%JAK2V617F) decreased from 37% (range: 1.9-95) at baseline to 22.5% (range: 0-85) at 24 months. In pts achieving CR or PR (n=7), the reduction in %JAK2V617F was greater (median: 42% to 18%, p&lt;0.05) compared with pts achieving NR (n=12) (median: 30% to 22%, p&lt;0.005). Stratified according to molecular response (MR) (n=4) or non-MR (n=13), median % JAK2V617F at baseline was 40 (21-95) and 43 (42-70) in non-MR and MR, respectively, and 31 (11-85) and 13 (1.8-25) in non-MR and MR, respectively during treatment. At baseline, pts achieving MR had no non-driver mut and pts achieving non-MR had a median number of non-driver mut of 1 (range: 0-7) (p&lt;0.02). Eight pts were not evaluable because they were either CALR or MPL positive, triple-negative, or had a baseline JAK2V617F ≤ 20%. In all pts, there were 30 non-driver mut in 15 genes at baseline and 40 non-driver mut in 19 genes at 24 months of therapy. At baseline and at 24 months, TP53 occurred in 6 and 6 (24%), TET2, ASXL1, RUNX1, or SF3B1 in 2 and 3 (8/12%), and CBL, DNMT3A, or SRSF2 in 2 and 2 (8%) pts, respectively. Eleven new non-driver post-treatment mut occurred in 9 pts and were more prevalent in pts with MF (6/9, 66%) compared to pts with PV (3/16, 19%), p&lt;0.02. However, the VAF was low (median 1.2%, range 1.02% - 3.6%) and none of the new 11 post-treatment mut appeared in the same gene. Discussion and Conclusions: Earlier studies have shown an association between poor response to IFN and number of mut or high molecular risk mut. In this study, the presence of non-driver mut was associated with a significantly poorer molecular response. However, there was no association between the presence or type of non-driver mut and clinico-hematologic response. At 24 months, there was an evolution of subclones with a significantly higher number of new subclones appearing in pts with MF compared to pts with PV. However, the VAF was low and none of the acquired mut appeared in the same gene contradictory to another study showing an association between treatment-emergent mut in DNMT3A and treatment with IFN. In conclusion, these data show that a clinico-hematologic response is achievable during combination therapy despite the presence of non-driver mutations at baseline. Disclosures Bjørn: Novartis: Other: Ruxolitnib. Hasselbalch: Novartis, AOP Orphan: Consultancy, Other: Advisory Board. OffLabel Disclosure: Interferon-alpha for treatment

Utility of somatic mutation panel testing in patients with advanced cancer receiving treatment in an Irish teaching hospital.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 3076-3076
Author(s):  
Hadia Khan ◽  
Louise O' Callaghan ◽  
Gul Ahmed ◽  
Brian Richard Bird ◽  
Derbrenn O'Connor ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Somatic Mutation ◽  
Teaching Hospital ◽  
Somatic Mutations ◽  
Tumour Response ◽  
Standard Of Care ◽  
Genetic Alterations ◽  
Panel Testing ◽  
The Mean ◽  
The Impact

3076 Background: Tumor testing for potentially actionable somatic mutations via commercially available panel tests has entered routine clinical practice in many countries. In Ireland the cost of these tests is not covered by insurance companies and must be paid for by patients. Use of these tests is sporadic and depends on patient and clinician factors (including ability to pay). Existing data suggest that such testing results in a direct impact on patient therapy in a minority of patients only. We reviewed the impact of somatic mutation testing on treatment selection and outcomes in patients attending a medical oncology service in a teaching hospital in Ireland. Methods: We performed a retrospective study of patients who had commercial panel testing performed as part of routine oncology care. All patients opportunistically tested between 2013 and 2018 were included. Patients having focused molecular tests for approved therapies (e.g. RAS mutations in colon cancer, EGFR and ALK mutations in non-small cell lung cancer) were excluded.We reviewed medical records to assess the frequency and utility of mutations detected, the impact of testing on next and subsequent lines of therapy, and the effectiveness of therapy. Results: 74 panel tests were performed in 71 patients. 39 tests (53%) detected mutations, of which 21 (28%) were potentially actionable. 36 patients (51%) had further treatment after testing was performed. 9 tests (12%) led to test-based treatment. The mean duration of test-based treatment was 34 days (range 1-90 days). No patients had benefit from test based treatment, defined as tumour response or disease stabilisation on restaging scans. 23 patients died within 90 days of panel tests being requested. Among patients starting and completing a subsequent line of therapy after testing, the mean duration of therapy with test-based treatment was 39 days (range 6-90) and for standard of care treatment was 56 days (range 1-262 days). Conclusions: While testing for tumor-specific somatic mutations with proven predictive benefit is very useful, somatic mutation panel testing for non standard of care genetic alterations is not of utility in this real world setting. Its role in Ireland should be limited to identification of suitable early phase clinical trials. Discussions of panel testing should include frank discussion of expected benefits, and should also address factors such as patient ability to travel for clinical trials. [Table: see text]

scholarly journals The role of cytogenetic abnormalities as a prognostic marker in primary myelofibrosis: applicability at the time of diagnosis and later during disease course

Blood ◽  
2009 ◽  
Vol 113 (18) ◽  
pp. 4171-4178 ◽  
Author(s):  
Constantine S. Tam ◽  
Lynne V. Abruzzo ◽  
Katherine I. Lin ◽  
Jorge Cortes ◽  
Alice Lynn ◽  
...  
Keyword(s):  
Median Survival ◽  
Prognostic Significance ◽  
Chronic Phase ◽  
Primary Myelofibrosis ◽  
Prognostic Scores ◽  
Chromosome 17 ◽  
Disease Course ◽  
Cytogenetic Abnormalities ◽  
The Impact

Abstract Although cytogenetic abnormalities are important prognostic factors in myeloid malignancies, they are not included in current prognostic scores for primary myelofibrosis (PMF). To determine their relevance in PMF, we retrospectively examined the impact of cytogenetic abnormalities and karyotypic evolution on the outcome of 256 patients. Baseline cytogenetic status impacted significantly on survival: patients with favorable abnormalities (sole deletions in 13q or 20q, or trisomy 9 ± one other abnormality) had survivals similar to those with normal diploid karyotypes (median, 63 and 46 months, respectively), whereas patients with unfavorable abnormalities (rearrangement of chromosome 5 or 7, or ≥ 3 abnormalities) had a poor median survival of 15 months. Patients with abnormalities of chromosome 17 had a median survival of only 5 months. A model containing karyotypic abnormalities, hemoglobin, platelet count, and performance status effectively risk-stratified patients at initial evaluation. Among 73 patients assessable for clonal evolution during stable chronic phase, those who developed unfavorable or chromosome 17 abnormalities had median survivals of 18 and 9 months, respectively, suggesting the potential role of cytogenetics as a risk factor applicable at any time in the disease course. Dynamic prognostic significance of cytogenetic abnormalities in PMF should be further prospectively evaluated.

scholarly journals The genetic basis of myelodysplasia and its clinical relevance

Blood ◽  
2013 ◽  
Vol 122 (25) ◽  
pp. 4021-4034 ◽  
Author(s):  
Mario Cazzola ◽  
Matteo G. Della Porta ◽  
Luca Malcovati
Keyword(s):  
Rna Splicing ◽  
Prognostic Significance ◽  
Chromatin Modification ◽  
Normal Karyotype ◽  
Driver Mutations ◽  
Refractory Anemia ◽  
Diagnostic Feature ◽  
Driver Genes ◽  
Myeloid Neoplasms ◽  
Oncogenic Mutations

Abstract Myelodysplasia is a diagnostic feature of myelodysplastic syndromes (MDSs) but is also found in other myeloid neoplasms. Its molecular basis has been recently elucidated by means of massive parallel sequencing studies. About 90% of MDS patients carry ≥1 oncogenic mutations, and two thirds of them are found in individuals with a normal karyotype. Driver mutant genes include those of RNA splicing (SF3B1, SRSF2, U2AF1, and ZRSR2), DNA methylation (TET2, DNMT3A, and IDH1/2), chromatin modification (ASXL1 and EZH2), transcription regulation (RUNX1), DNA repair (TP53), signal transduction (CBL, NRAS, and KRAS), and cohesin complex (STAG2). Only 4 to 6 genes are consistently mutated in ≥10% MDS patients, whereas a long tail of ∼50 genes are mutated less frequently. At presentation, most patients typically have 2 or 3 driver oncogenic mutations and hundreds of background mutations. MDS driver genes are also frequently mutated in other myeloid neoplasms. Reliable genotype/phenotype relationships include the association of the SF3B1 mutation with refractory anemia with ring sideroblasts, TET2/SRSF2 comutation with chronic myelomonocytic leukemia, and activating CSF3R mutation with chronic neutrophilic leukemia. Although both founding and subclonal driver mutations have been shown to have prognostic significance, prospective clinical trials that include the molecular characterization of the patient’s genome are now needed.

scholarly journals Dual Deep Sequencing Improves the Accuracy of Low-Frequency Somatic Mutation Detection in Cancer Gene Panel Testing

2020 ◽  
Vol 21 (10) ◽  
pp. 3530
Author(s):  
Hiroki Ura ◽  
Sumihito Togi ◽  
Yo Niida
Keyword(s):  
Somatic Mutations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Low Frequency ◽  
Gene Panel ◽  
Missense Mutations ◽  
Cancer Gene ◽  
Single Nucleotide Variants ◽  
Somatic Variant ◽  
Panel Testing ◽  
Gene Panel Testing

Cancer gene panel testing requires accurate detection of somatic mosaic mutations, as the test sample consists of a mixture of cancer cells and normal cells; each minor clone in the tumor also has different somatic mutations. Several studies have shown that the different types of software used for variant calling for next generation sequencing (NGS) can detect low-frequency somatic mutations. However, the accuracy of these somatic variant callers is unknown. We performed cancer gene panel testing in duplicate experiments using three different high-fidelity DNA polymerases in pre-capture amplification steps and analyzed by three different variant callers, Strelka2, Mutect2, and LoFreq. We selected six somatic variants that were detected in both experiments with more than two polymerases and by at least one variant caller. Among them, five single nucleotide variants were verified by CEL nuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) and Sanger sequencing. In silico analysis indicated that the FBXW7 and MAP3K1 missense mutations cause damage at the protein level. Comparing three somatic variant callers, we found that Strelka2 detected more variants than Mutect2 and LoFreq. We conclude that dual sequencing with Strelka2 analysis is useful for detection of accurate somatic mutations in cancer gene panel testing.

scholarly journals The Diagnostic Utility of Mutational Analysis in Patients with Suspected Myeloid Neoplasms, a Single Institution Experience

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5545-5545
Author(s):  
Jason Macklis ◽  
Jamile M. Shammo ◽  
Lela Buckingham ◽  
Parameswaran Venugopal
Keyword(s):  
Research Funding ◽  
Prognostic Significance ◽  
Genetic Alterations ◽  
Low Risk ◽  
Jak2 Inhibitor ◽  
Molecular Abnormalities ◽  
Panel Testing ◽  
Transplant Evaluation ◽  
The Impact ◽  
Calr Mutation

Abstract Introduction Molecular abnormalities in primary myeloid diseases have been well described. Some of these molecular alterations are nearly pathognomonic for specific diseases such as JAK2 V617F and CALR, whereas others are more generally associated with myeloid disorders, though they may have prognostic significance. Commercially available panel testing screen for multiple genetic alterations simultaneously and are used increasingly for diagnostic and prognostic purposes. We sought to evaluate the clinical utility of panel testing in primary myeloid disorders and to evaluate the impact on diagnosis and management. Methods Patients who presented to the MDS/MPN clinic between February, 2015 and May, 2016 were included in the study if a Genoptix Myeloid Molecular Panel (MMP) was submitted and resulted. The MMP tests for 40 recurrently mutated genes in myeloid malignancies using next generation sequencing. It includes genes involved with RNA splicing, epigenetics, transcription factors, cohesion complex, activated signaling, and others. Primary diagnosis (if determined), and rationale for testing (diagnostic, diagnosis in question, or prognostic) at the time the test was ordered was recorded. Charts were reviewed to determine if a diagnosis was made or if there was a change in diagnosis, prognosis, or management as a result of the MMP. Results 40 patients had a resulted MMP. The primary diagnoses at presentation were: ICUS (8), MDS (18), MF (7), CMML (2), MPN-U (2), MDS/MPN (1), and multiple diagnoses (2). Of the 40 patients that were evaluated, 35 patients had at least one reported mutation. 5 MMPs (12.5%) resulted in a change in diagnosis. 2 patients diagnosed with MPN-U at presentation were found to carry CSF3R mutation consistent with a diagnosis of chronic neutrophilic leukemia. 3 additional patients had an original diagnosis of MDS with fibrosis; however, 2 of these patients had a MPL mutation and third had a CALR mutation consistent with myelofibrosis. Of prognostic significance, 5 of the 18 patients with MDS (27.8%) were found to harbor TP53 mutations. Of these, 4 (22.2%) patients were previously classified as having low risk disease by IPSS, 2 (11.1%) of which had deletion 5q as their only cytogenetic abnormality. Altogether, 8 (20%) patients had a drastic change in diagnosis or prognosis. In 4 (10%) patients, this resulted in a change in management as follows: of the 2 patients that had a diagnosis of CNL, 1 was enrolled on a clinical trial involving a JAK2 inhibitor; 1 of the 2 patients with a history of MDS changed to MF by the identification of a CALR mutation was treated with a JAK2 inhibitor; and 2 of the patients with low risk MDS were referred for transplant evaluation due to the presence of a TP53 mutation where transplant would otherwise not be indicated. Conclusions: In this small series, we find that the MMP can aid in making an accurate diagnosis and has prognostic significance particularly in the following patient populations: MDS with fibrosis, MPN-U, and young patients with low-risk MDS who may benefit from closer monitoring and early referral for transplant evaluation. Confirmation of these observations merits prospective evaluation for a larger number of patients. Disclosures Shammo: novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; incyte: Consultancy, Honoraria, Research Funding, Speakers Bureau; alexion: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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